Content determination and identification method for Shenxianling granules

Abstract

The invention relates to the technical field of detection, in particular to a content determination and identification method for Shenxianling granules, which comprises the following steps: determining the content of oridonin in the Shenxianling granules by adopting a high performance liquid chromatography (HPLC), and respectively identifying six components, namely hairyvein agrimony, dark plum, liquorice, gynostemma pentaphyllum, ginseng and lucid ganoderma by adopting a thin layer chromatography (TLC). The TLC identification method of each component comprises a unique preparation method of a test solution, a control medicinal material solution and a negative control solution, and specific setting of a sample application amount, a developing solvent and an inspection condition; according to the invention, a full-chain quality control system of the Shenxianling granules is constructed through the technical combination of accurate quantification of characteristic components, exclusive qualitative determination of multiple medicines and efficient removal of impurities. According to the HPLC method, the oridonin is accurately quantified, and the problem that original standard indexes are one-sided is solved; the TLC identification system ensures rapid true and false identification of the six traditional Chinese medicines through double optimization of extraction conditions and an expansion system.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for content determination and identification of Shenxianling granules. Background Art

[0002] Shenxianling Granules, a compound preparation composed of eight traditional Chinese medicines, including ginseng, Rubescens herb, and Agrimoniae, have the effects of replenishing qi and promoting body fluid, clearing away heat and detoxifying, and are widely used in adjuvant cancer treatment. The accuracy of its quality control is directly related to clinical efficacy and safety. However, the current quality standards have significant technical bottlenecks:

[0003] One-sidedness of content determination: only ginsenoside Rg1 in ginseng was used as the quantitative indicator, but the amount of ginseng used in the prescription was only 1 / 3 of that of Rubescens, and the content of Rubescensine A, the active ingredient with the highest content in the prescription, was not determined, resulting in the inability to fully reflect the quality of the preparation.

[0004] Lack of identification methods: There is a lack of exclusive identification methods for six Chinese medicinal materials, including herbaceous agrimony, black plum, liquorice, gynostemma pentaphyllum, and ginseng. The existing standards only rely on thin layer identification of ginseng, which makes it difficult to achieve full coverage quality control of the eight Chinese medicinal materials, and there is a risk of difficulty in distinguishing the authenticity and quality.

[0005] Impurity interference problem: The ingredients of compound preparations are complex, and traditional extraction methods are prone to introduce impurities such as pigments and polysaccharides, which interfere with the accuracy of content determination results. In addition, the current impurity removal process has not been optimized for the characteristics of oridonin, affecting the detection sensitivity.

[0006] Inefficient quality control: There is a lack of a systematic testing system for the main active ingredients in the preparations, which cannot meet the requirements of the "Chinese Pharmacopoeia" for Chinese patent medicines to be "safe, effective, and quality-controlled". In particular, there are technical gaps in batch stability evaluation and production process optimization.

[0007] To address the above problems, the present invention established a high-performance liquid chromatography (HPLC) content determination method for Rubescensine A, combined with a thin-layer chromatography (TLC) identification system for six traditional Chinese medicines, and constructed a quality control technology covering the entire prescription through a composite impurity removal process and exclusive detection condition optimization, filling the gap in existing standards. Summary of the Invention

[0008] The present invention aims to provide an HPLC method for determining the content of oridonin in Shenxianling Granules, a traditional Chinese medicine used for the treatment of digestive tract tumors, and to provide TLC identification of six Chinese medicinal ingredients: Agrimoniae, Prunus mume, Licorice, Prunus mume, Gynostemma pentaphyllum, and Panax ginseng. The HPLC method for determining the content of oridonin in Shenxianling Granules can be used to investigate the stability of Shenxianling Granules. This method is easy to operate, highly specific, has good resolution, high stability, and is highly efficient. It can also simultaneously determine seven ingredients, effectively monitoring product quality.

[0009] The present invention provides the following technical solutions:

[0010] A method for content determination and identification of Shenxianling granules comprises determining the content of oridonin in Shenxianling granules by high performance liquid chromatography (HPLC), and identifying six ingredients, namely, herba agrimonii, plums, licorice, gynostemma pentaphyllum, ginseng, and ganoderma lucidum, by thin layer chromatography (TLC); the TLC identification method for each ingredient comprises a unique preparation method for a test solution, a control medicinal material solution, and a negative control solution, as well as specific settings for the sample spotting amount, developing agent, and inspection conditions.

[0011] As a further technical solution, the chromatographic conditions for the HPLC method for determining the content of Rubescensine A are: Phenomenon C18 chromatographic column (250mm×4.6μm, 5μm); mobile phase is methanol:water = 65:35; flow rate 0.8-1.0ml / min; column temperature 25-26°C; detection wavelength 238nm; injection volume 20μl.

[0012] As a further technical solution, the preparation and pretreatment method of the test solution is as follows: grind the Shenxianling granules, weigh 15-16g and place it in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60-90°C for 2.5h, discard the petroleum ether, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, ultrasonically treat for 20-30min, pour out the methanol extract, and then use 20ml of methanol to operate 5 times in the same way, combine the methanol extracts and evaporate to dryness, and dilute the residue to a 50ml volumetric flask with a solution of methanol: water = 65:35, add 1.5-1.8g of a composite impurity remover, shake for 15-20min, filter, and filter the filtrate with a 0.4μm microporous filter membrane to obtain the test solution;

[0013] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a cross-linking-calcination process;

[0014] The composite impurity remover is prepared by mixing silica gel (particle size 50-100 μm), neutral alumina, and activated carbon in a mass ratio of 3-5:2-3:1-2, adding a 5-6% polyvinyl alcohol aqueous solution as a cross-linking agent, stirring at 60° C. into a paste, extruding into particles with a diameter of 1-2 mm, calcining in a muffle furnace at 400-430° C. for 2-3 hours, and filtering through an 80-mesh sieve after cooling for later use;

[0015] The mass fraction of the polyvinyl alcohol aqueous solution is 20%.

[0016] As a further technical solution, the preparation method of the reference solution is as follows: accurately weigh 8 mg of oridonin standard, add 4 ml of methanol to dissolve it to prepare a oridonin stock solution with a concentration of 2 mg / ml; when preparing the standard curve, dilute the oridonin stock solution with methanol to reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, and 0.78 μg / ml, respectively; divide the solution into 1.5 ml centrifuge tubes, and store at 4°C for future use.

[0017] As a further technical solution, when the TLC method is used to identify the components of Agrimoniae:

[0018] Preparation of test solution: Take 5 g of Agrimony Root, add 60 ml of petroleum ether (60-90 ℃) and ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of chloroform, shake and extract with 10 ml of 5% sodium hydroxide solution, discard the chloroform solution, adjust the pH value of the sodium hydroxide solution to 1-2 with dilute hydrochloric acid, and then shake and extract with chloroform twice (10 ml each time), combine the chloroform solutions, add 10 ml of water to wash, discard the water solution, and concentrate the chloroform solution to 1 ml;

[0019] Control system: 5 g of Agrimoniae negative preparation and 0.5 g of Agrimoniae control medicinal material were used to prepare negative control solution and control medicinal material solution in the same manner;

[0020] Spotting and development: Pipette 10 μl each of the test solution, negative control solution and control medicinal material solution, spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of petroleum ether (60-90℃)-ethyl acetate-acetic acid (100:9:5) as the developing agent.

[0021] As a further technical solution, when the TLC method is used to identify the components of ebony:

[0022] Preparation of test solution: Take 15g of the powder of this product, add 40ml of methanol and sonicate for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, add ether and shake to extract twice (20ml each time), combine the ether solution and evaporate to dryness, soak the residue in petroleum ether (30-60℃) twice (20ml each time), pour off the petroleum ether, and dissolve the residue in 1ml of anhydrous ethanol;

[0023] Control system: 15g of Wumei negative preparation and 1g of Wumei control medicinal material. Prepare negative control solution and control medicinal material solution in the same way;

[0024] Spotting and development: 5 μl of the test solution, 5 μl of the negative control solution, and 10 μl of the control medicinal material solution were applied to the same silica gel G thin layer plate and developed with cyclohexane-chloroformane-ethyl acetate-formic acid (20:5:8:0.1) as the developing solvent.

[0025] As a further technical solution, when the TLC method is used to identify the components of licorice:

[0026] Preparation of test solution: Take 15g of the powder of this product, add 60ml of ether and heat under reflux for 1h, filter and discard the ether solution, add 60ml of methanol to the residue and heat under reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, extract with n-butanol 3 times (20ml each time), combine the n-butanol solutions, wash 3 times with water, discard the water solution, evaporate the n-butanol solution to dryness, and dissolve the residue in 1ml of methanol;

[0027] Control system: 15g of negative preparation lacking licorice and 1g of licorice control medicinal material were used to prepare negative control solution and control medicinal material solution in the same way;

[0028] Spotting and development: Pipette 10 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, and develop with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing agent.

[0029] As a further technical solution, when the TLC method is used to identify the components of Gynostemma pentaphyllum:

[0030] Preparation of test solution: Take 15g of the powder of this product, add 60ml of methanol and sonicate for 1h, cool and filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract with saturated n-butanol 3 times (20ml each time), combine the n-butanol solutions and evaporate to dryness, dissolve the residue in 1ml of methanol;

[0031] Control system: 15 g of the negative preparation lacking Gynostemma pentaphyllum and 1 g of the Gynostemma pentaphyllum control medicinal material were used to prepare the negative control solution and the control medicinal material solution in the same manner;

[0032] Spotting and development: Pipette 5μl of the test solution and negative control solution, and 10μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of n-butanol-ethyl acetate-water (4:1:5) placed below 10℃ as the developing agent.

[0033] As a further technical solution, when the TLC method is used to identify ginseng components:

[0034] Preparation of test solution: Take 15g of the powder of this product, add 100ml of chloroform and heat under reflux for 1h, discard the chloroform solution, evaporate the solvent from the residue, add 1.0ml of water and stir to moisten it, add 30ml of water-saturated n-butanol and ultrasonicate for 1h, aspirate the supernatant, add 3 times the amount of ammonia test solution, shake well and let it separate, take the upper layer and evaporate to dryness, and dissolve the residue in 1ml of methanol;

[0035] Control system: 15g of ginseng negative preparation and 1g of ginseng control medicinal material were used to prepare negative control solution and control medicinal material solution in the same way;

[0036] Spotting and development: Pipette 10 μl each of the test solution, negative control solution, and control medicinal material solution, and spot them on the same silica gel G thin layer plate. Develop with the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) stored below 10°C as the developing agent.

[0037] As a further technical solution, when the TLC method is used to identify the components, the samples must be air-dried after development, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and inspected under ultraviolet light (365nm); in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, and there are no spots at the corresponding positions in the negative control solution.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention achieves accurate and comprehensive quality control of Shenxianling granules through multi-dimensional technological innovation, as follows:

[0040] 1. Improved accuracy of HPLC content determination

[0041] (I) Separation mechanism of chromatographic condition optimization

[0042] Selection of stationary phase: A Phenylephrine C18 column (250 mm × 4.6 μm, 5 μm) was used. Its octadecyl bonded phase forms specific retention with Rubescensine A molecules through hydrophobic interaction. Combined with a methanol-water (65:35) mobile phase system, the separation degree between the target peak and the impurity peak reached 2.8 (standard ≥1.5), effectively eliminating the interference of the compound matrix.

[0043] (2) Mechanism of action of composite impurity remover

[0044] Multi-component synergistic purification: Silica gel (adsorbs pigments and sterols), neutral alumina (captures acidic impurities), and activated carbon (adsorbs polyphenols) are combined in a specific mass ratio to selectively remove interfering components through a dual mechanism of physical adsorption and chemical action. Silica gel has an adsorption capacity of 50mg / g for polar impurities, and activated carbon has a removal rate of over 90% for phenolic acids that absorb in the ultraviolet region.

[0045] Structural optimization enhances performance: After cross-linking with polyvinyl alcohol and high-temperature calcination, the composite impurity remover forms a porous structure with uniform pore size distribution, which greatly increases the specific surface area, significantly improving the impurity adsorption efficiency compared to single silica gel, while avoiding excessive adsorption of oridonin.

[0046] 2. Breakthrough in the Specificity of the TLC Identification System

[0047] (I) Targeted extraction of test solution preparation

[0048] Polarity gradient separation: Design a dedicated extraction process based on the solubility differences of different components:

[0049] Agrimony Root (non-polar component): After ultrasonic extraction with petroleum ether, phenolic acid components were enriched by NaOH-hydrochloric acid back extraction. The concentration of the target component increased by 5 times after the chloroform layer was concentrated.

[0050] Licorice (polar saponins): After degreasing with ether, methanol reflux extraction and extraction with n-butanol, the purity of glycyrrhizic acid reaches more than 95%, eliminating the interference of fat-soluble impurities.

[0051] Acid-base environment regulation: For example, in the identification of black plums, the concentration of black plum acid in the test solution is increased to 2 mg / ml through step-by-step degreasing with ether and petroleum ether and dissolution with anhydrous ethanol, which is 2.5 times higher than that of the direct extraction method, and the clarity of the spot color development is significantly enhanced.

[0052] (2) Adaptation of developing agent and inspection conditions

[0053] Selective separation mechanism:

[0054] Ginseng identification uses a lower layer developing solvent of chloroform-ethyl acetate-methanol-water (15:40:22:10), taking advantage of the polarity difference of ginsenoside Rg1 (logP=2.3), so that it is separated from other saponins by a distance of 8 cm, thereby improving the spot recognition.

[0055] The identification of Gynostemma pentaphyllum was carried out using the upper layer solution of n-butanol-ethyl acetate-water (4:1:5) as the developing solvent. By adjusting the proportion of the aqueous phase (50%), the Rf value of gypenosapogenin was controlled at 0.4-0.6, with no cross-interference with the negative control.

[0056] Enhanced fluorescence color development: After spraying with 10% sulfuric acid ethanol solution and heating at 105°C, the characteristic spots of each component show unique fluorescence under 365nm ultraviolet light (such as glycyrrhizic acid shows yellow-green fluorescence), and the detection sensitivity reaches μg level, which is significantly improved compared with visible light inspection.

[0057] The present invention constructs a full-chain quality control system for Shenxianling Granules through the technical combination of "precise quantification of characteristic components + exclusive qualitative analysis of multiple medicinal flavors + efficient removal of impurities." Among them, the HPLC method achieves accurate quantification of Rubescensine A, solving the problem of one-sided indicators in the original standard; the TLC identification system ensures rapid authenticity determination of the six traditional Chinese medicines through dual optimization of extraction conditions and development systems; and the composite impurity removal technology improves the reliability of test data from the source. This solution upgrades the quality control of preparations from "single indicator monitoring" to "full component coverage assessment," which not only meets the latest requirements of the "Chinese Pharmacopoeia" for the quality standards of traditional Chinese medicines, but also provides a technical paradigm that can be used as a reference for improving the quality of similar compound preparations, and has significant clinical application value and industrial promotion significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a curve chart showing the effect of calcination temperature of different composite adsorbents on their polysaccharide removal rate. DETAILED DESCRIPTION

[0059] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0060] The present invention provides a method for determining and identifying the content of Shenxianling granules, comprising the following steps:

[0061] High performance liquid chromatography (HPLC) was used to determine the content of oridonin in Shenxianling granules, and thin layer chromatography (TLC) was used to identify six ingredients, including herb agrimony, plum, liquorice, Gynostemma pentaphyllum, ginseng, and Ganoderma lucidum. The TLC identification method for each ingredient included the preparation of test solution, control herb solution, and negative control solution, as well as the specific settings of sample volume, developing agent, and inspection conditions.

[0062] In the present invention, the chromatographic conditions for determining the content of Rubescensine A by HPLC are as follows: Phenomenon C18 chromatographic column (250 mm × 4.6 μm, 5 μm); mobile phase of methanol: water = 65:35; flow rate 0.8-1.0 ml / min; column temperature 25-26 ° C; detection wavelength 238 nm; injection volume 20 μl.

[0063] In the present invention, the preparation and pretreatment method of the test solution is as follows: grind the Shenxianling granules, weigh 15-16g and place them in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60-90°C for 2.5h, discard the petroleum ether, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, ultrasonically treat for 20-30min, pour out the methanol extract, and then use 20ml of methanol to operate 5 times in the same way, combine the methanol extracts and evaporate to dryness, dilute the residue to a 50ml volumetric flask with a solution of methanol: water = 65:35, add 1.5-1.8g of a composite impurity remover, and shake. Shake for 15-20 minutes, filter, and take the filtrate and filter it with a 0.4μm microporous filter membrane to obtain the test solution; the composite impurity remover is prepared by a crosslinking-calcination process of silica gel-alumina-activated carbon, and its preparation method is as follows: silica gel (particle size 50-100μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3-5:2-3:1-2, 5-6% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, stirred into a paste at 60°C, extruded into particles with a diameter of 1-2mm, calcined at 400-430°C in a muffle furnace for 2-3h, cooled, and passed through an 80-mesh sieve for use.

[0064] In the present invention, the preparation method of the reference solution is as follows: accurately weigh 8 mg of oridonin standard, add 4 ml of methanol to dissolve it to prepare a oridonin stock solution with a concentration of 2 mg / ml; when preparing the standard curve, dilute the oridonin stock solution with methanol to reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, and 0.78 μg / ml, respectively; divide the solution into 1.5 ml centrifuge tubes, and store at 4°C for future use.

[0065] In the present invention, the TLC method is used to identify the components of Agrimoniae: the test solution is prepared by taking 5g of Agrimoniae, adding 60ml of petroleum ether (60-90°C) and ultrasonically treating it for 1h, filtering, evaporating the filtrate to dryness, dissolving the residue in 10ml of chloroform, and shaking and extracting it with 10ml of 5% sodium hydroxide solution. The chloroform solution is discarded, and the pH value of the sodium hydroxide solution is adjusted to 1-2 with dilute hydrochloric acid, and then shaking and extracting it with chloroform twice (10ml each time), and the chloroform solutions are combined. Add 10 ml of water for washing, discard the water, and concentrate the chloroform solution to 1 ml; the control system is 5 g of the negative preparation lacking Agrimoniae and 0.5 g of Agrimoniae control medicinal material. The negative control solution and control medicinal material solution are prepared in the same way; the spotting and development are to absorb 10 μl of the test solution, negative control solution and control medicinal material solution respectively, spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of petroleum ether (60-90℃)-ethyl acetate-acetic acid (100:9:5) as the developing agent.

[0066] In the present invention, when the TLC method is used to identify the components of black plum, the test solution is prepared by taking 15 g of the powder of the product, adding 40 ml of methanol and ultrasonically treating it for 1 hour, filtering, evaporating the filtrate to dryness, dissolving the residue in 40 ml of water, adding ether and shaking to extract twice (20 ml each time), combining the ether solutions and evaporating to dryness, soaking the residue in petroleum ether (30-60°C) twice (20 ml each time), decanting the petroleum ether, and dissolving the residue in 1 ml of anhydrous ethanol; the control system is 15 g of a negative preparation lacking black plum and 1 g of a black plum control medicinal material, and a negative control solution and a control medicinal material solution are prepared in the same manner; spotting and development are performed by aspirating 5 μl of the test solution, 5 μl of the negative control solution, and 10 μl of the control medicinal material solution, and spotting them on the same silica gel G thin layer plate, and developing with cyclohexane-chloroform-ethyl acetate-formic acid (20:5:8:0.1) as a developing solvent.

[0067] In the present invention, when the TLC method is used to identify licorice components: the test solution is prepared by taking 15g of the product powder, adding 60ml of ether and heating under reflux for 1h, filtering, and discarding the ether solution, adding 60ml of methanol to the medicinal residue and heating under reflux for 1h, filtering, and evaporating the filtrate to dryness, dissolving the residue in 40ml of water, and extracting it three times with n-butanol (20ml each time), combining the n-butanol solutions, washing it three times with water, and discarding the water solution, evaporating the n-butanol solution to dryness, and dissolving the residue in 1ml of methanol; the control system is 15g of a negative preparation lacking licorice and 1g of a licorice control medicinal material, and a negative control solution and a control medicinal material solution are prepared in the same manner; spotting and development are as follows: aspirating 10μl of each of the above solutions, spotting them on the same silica gel G thin layer plate, and developing with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as a developing solvent.

[0068] In the present invention, when the TLC method is used to identify the components of Gynostemma pentaphyllum, the test solution is prepared by taking 15 g of the powder of the product, adding 60 ml of methanol for ultrasonic treatment for 1 hour, cooling and filtering, evaporating the filtrate to dryness, dissolving the residue in 20 ml of water, extracting it three times with water-saturated n-butanol (20 ml each time), combining the n-butanol solutions and evaporating them to dryness, and dissolving the residue in 1 ml of methanol; the control system is 15 g of a negative preparation lacking Gynostemma pentaphyllum and 1 g of a Gynostemma pentaphyllum control medicinal material, and a negative control solution and a control medicinal material solution are prepared in the same manner; spotting and development are performed by aspirating 5 μl of the test solution and the negative control solution, and 10 μl of the control medicinal material solution, and spotting them on the same silica gel G thin layer plate, and developing with an upper layer solution of n-butanol-ethyl acetate-water (4:1:5) placed below 10°C as a developing agent.

[0069] In the present invention, when the TLC method is used to identify ginseng components: the test solution is prepared by taking 15g of the product powder, adding 100ml of chloroform and heating under reflux for 1h, discarding the chloroform liquid, evaporating the solvent from the medicinal residue, adding 1.0ml of water and stirring to moisten it, adding 30ml of water-saturated n-butanol and ultrasonically treating it for 1h, absorbing the supernatant, adding 3 times the amount of ammonia test solution, shaking and standing to separate the layers, taking the upper layer liquid and evaporating it to dryness, and dissolving the residue in 1ml of methanol; the control system is 15g of the ginseng-deficient negative preparation and 1g of ginseng control medicinal material, and the negative control solution and control medicinal material solution are prepared in the same way; the sample spotting and development are as follows: 10μl of the test solution, negative control solution, and control medicinal material solution are each aspirated and spotted on the same silica gel G thin layer plate, and the lower layer solution placed below 10°C in chloroform-ethyl acetate-methanol-water (15:40:22:10) is used as the developing agent.

[0070] In the present invention, when the TLC method is used to identify each component, the sample must be air-dried after development, sprayed with a 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and inspected under an ultraviolet lamp (365nm); in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, and there are no spots at the corresponding positions in the negative control solution.

[0071] The materials and equipment used in the present invention are all commercially available products in the art.

[0072] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0073] Example 1

[0074] HPLC determination: Grind the Shenxianling granules into powder, weigh 15g and place it in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60℃ for extraction for 2.5h, discard the petroleum ether, evaporate it, add 30ml of methanol and soak for 2h, ultrasonicate for 20min, use 20ml of methanol to operate in the same way 5 times, combine the methanol extracts and evaporate to dryness, make up the volume of the residue to a 50ml volumetric flask with a solution of methanol: water = 65:35, add 1.5g of a composite impurity remover, shake for 15min, filter and take the filtrate and filter it with a 0.4μm microporous filter membrane to obtain the test solution; according to HPLC chromatographic conditions, the concentration of oridonin was measured to be 85μg / ml.

[0075] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a crosslinking-calcination process. The preparation method is as follows: silica gel (particle size 100 μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3:2:1, a 5% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, and the mixture is stirred into a paste at 60° C., extruded into particles with a diameter of 1 mm, calcined in a muffle furnace at 400° C. for 2 hours, and passed through an 80-mesh sieve after cooling for later use.

[0076] Identification of Agrimony by TLC: Prepare the test sample solution, negative control solution and control medicinal material solution according to the above-mentioned Agrimony identification method. After spotting and development, the test sample chromatogram and the control medicinal material chromatogram show the same blue-purple fluorescent spots at the corresponding positions, while there are no spots at the corresponding positions of the negative control solution.

[0077] Example 2

[0078] HPLC determination: 15.5 g of Shenxianling granules were weighed, 1.6 g of the composite impurity remover was added, and the remaining operations were the same as in Example 1. The measured concentration of Rubescensine A was 88 μg / ml.

[0079] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a crosslinking-calcination process. The preparation method is as follows: silica gel (particle size 100 μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3:2:1, a 5% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, and the mixture is stirred into a paste at 60° C., extruded into particles with a diameter of 1 mm, calcined in a muffle furnace at 400° C. for 2 hours, and passed through an 80-mesh sieve after cooling for later use.

[0080] Identification of black plum by TLC: According to the above-mentioned identification method of black plum, the chromatogram of the test sample and the chromatogram of the control medicinal material show the same orange-yellow fluorescent spots at the corresponding positions, and there are no spots at the corresponding positions of the negative control solution.

[0081] Example 3

[0082] HPLC determination: 16 g of Shenxianling granules were weighed, 1.8 g of the composite impurity remover was added, and ultrasonic treatment was performed for 30 min. The remaining operations were the same as those in Example 1. The measured concentration of Rubescensine A was 90 μg / ml.

[0083] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a crosslinking-calcination process. The preparation method is as follows: silica gel (particle size 100 μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3:2:1, a 5% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, and the mixture is stirred into a paste at 60° C., extruded into particles with a diameter of 1 mm, calcined in a muffle furnace at 400° C. for 2 hours, and passed through an 80-mesh sieve after cooling for later use.

[0084] TLC identification of licorice: According to the above licorice identification method, the chromatogram of the test sample and the chromatogram of the control medicinal material show the same yellow-green fluorescent spots at the corresponding positions, and there are no spots at the corresponding positions of the negative control solution.

[0085] Example 4

[0086] HPLC determination: 15.2 g of Shenxianling granules were weighed, 1.7 g of the composite impurity remover was added, and ultrasonic treatment was performed for 22 min. The remaining operations were the same as those in Example 1. The measured concentration of Rubescensine A was 87 μg / ml.

[0087] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a crosslinking-calcination process. The preparation method is as follows: silica gel (particle size 100 μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3:2:1, a 5% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, and the mixture is stirred into a paste at 60° C., extruded into particles with a diameter of 1 mm, calcined in a muffle furnace at 400° C. for 2 hours, and passed through an 80-mesh sieve after cooling for later use.

[0088] TLC identification of Gynostemma pentaphyllum: According to the above-mentioned Gynostemma pentaphyllum identification method, the chromatogram of the test sample and the chromatogram of the control medicinal material show the same blue-green fluorescent spots at the corresponding positions, and there are no spots at the corresponding positions of the negative control solution.

[0089] Example 5

[0090] HPLC determination: 15.8 g of Shenxianling granules were weighed, 1.7 g of the composite impurity remover was added, and ultrasonic treatment was performed for 28 min. The remaining operations were the same as those in Example 1. The measured concentration of Rubescensine A was 89 μg / ml.

[0091] The composite impurity remover is prepared from silica gel-alumina-activated carbon through a crosslinking-calcination process. The preparation method is as follows: silica gel (particle size 100 μm), neutral alumina, and activated carbon are mixed in a mass ratio of 3:2:1, a 5% polyvinyl alcohol aqueous solution (mass fraction 20%) is added as a crosslinking agent, and the mixture is stirred into a paste at 60° C., extruded into particles with a diameter of 1 mm, calcined in a muffle furnace at 400° C. for 2 hours, and passed through an 80-mesh sieve after cooling for later use.

[0092] Identification of ginseng by TLC: According to the above ginseng identification method, the chromatogram of the test sample and the chromatogram of the control medicinal material show the same purple-red fluorescent spots at the corresponding positions, and there are no spots at the corresponding positions of the negative control solution.

[0093] Comparative Example 1

[0094] Treatment method: No composite impurity remover was added, and the remaining operations were the same as in Example 1.

[0095] Comparative Example 2

[0096] Treatment method: ginseng control medicinal material is missing, and the rest of the operations are the same as in Example 5.

[0097] Experimental verification

[0098] HPLC Methodology Validation

[0099] Experimental purpose: To verify the linearity, precision and recovery of the HPLC method for the determination of oridonin. Experimental method:

[0100] Linear relationship investigation: Take reference solution (concentrations are 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00 μg / ml, respectively), inject and measure according to HPLC chromatographic conditions, and draw a standard curve with peak area as the ordinate (Y) and concentration as the abscissa (X).

[0101] Precision test: Take the test solution of Example 2, inject it 6 times continuously, and record the peak area of Rubescensine A.

[0102] Sample addition recovery test: Take 10g of Shenxianling granule powder (Russobosine A content is 88μg / g) with a known content, a total of 6 portions, add 1ml of 100μg / ml Rubescensine A reference solution to each portion, treat according to the test solution preparation method, and determine and calculate the recovery rate.

[0103] Test data:

[0104] Table 1

[0105] Verification Project Test results Linear relationship Standard curve equation: Y=58234X+1256, R²=0.9998, linear range 0.78-100μg / ml. Precision The peak areas were 85623, 86125, 85897, 86012, 85784, and 85998, respectively, with RSD = 0.8% (n = 6). Sample recovery rate Measured values (μg / g): 175.2, 176.8, 174.9, 175.5, 176.1, 175.8, average recovery rate 99.7%, RSD=0.6%.

[0106] TLC identification specificity verification

[0107] Experimental purpose: To verify the specificity of TLC method for identifying ingredients such as Agrimony Root, Plum Blossom, and Licorice, and to eliminate negative interference. Experimental method:

[0108] Identification and verification of Agrimoniae: Take the test solution of Example 1, the negative control solution lacking Agrimoniae, and the control medicinal material solution of Agrimoniae, develop the samples according to TLC conditions, and observe the spot positions under ultraviolet light.

[0109] Identification and verification of black plum: Take the test solution of Example 2, the negative control solution lacking black plum, and the black plum control medicinal material solution, develop them according to TLC conditions, and observe the color and position of the spots.

[0110] Identification and verification of licorice: Take the test solution of Example 3, the negative control solution lacking licorice, and the licorice control medicinal material solution, develop them according to TLC conditions, and compare the chromatograms.

[0111] Test data:

[0112] Table 2

[0113] Element Test solution (Example) Negative control solution results Results of control medicinal material solution Agrimony Example 1 No fluorescent spots Blue-purple fluorescent spots Black plum Example 2 No fluorescent spots Orange-yellow fluorescent spots Licorice Example 3 No fluorescent spots Yellow-green fluorescent spots Gynostemma pentaphyllum Example 4 No fluorescent spots Blue-green fluorescent spots Ginseng Example 5 No fluorescent spots Purple-red fluorescent spots

[0114] Comparative Example 1: No composite impurity remover added

[0115] HPLC results: baseline noise was ±2 mAU, tailing factor of Rubescensine A peak was 1.8 (standard ≤1.2), and separation between impurity peak and target peak was 1.0 (standard ≥1.5).

[0116] TLC results: There were 5 spots in the chromatogram of the Herba Agrimoniae test sample (only 3 in the control herb), and there were 2 interfering spots in the negative control chromatogram at the corresponding positions.

[0117] Comparative Example 2: Lack of ginseng control medicinal materials

[0118] TLC results: The characteristic spots of ginsenosides were missing in the chromatogram of the test sample, and it could not be distinguished from the negative control, so the identification failed.

[0119] Reliability of HPLC method: good linear relationship, precision and recovery rate are in line with the requirements of the Chinese Pharmacopoeia (RSD≤2%, recovery rate 95%-105%), indicating that this method can accurately determine the content of oridonin.

[0120] TLC identification validity: The chromatograms of the test samples of each component correspond to the chromatograms of the reference medicinal materials, and there is no interference in the negative results, indicating that the TLC method has strong specificity and can be used for the qualitative identification of Shenxianling Granules.

[0121] Impact of key processes: The composite impurity remover is crucial for removing impurity interference. Without control herbs, identification cannot be completed, which verifies the necessity of the formula and process in the method of the present invention.

[0122] Impurity removal effect verification test

[0123] Test purpose: To verify the ability of the composite impurity remover to remove interfering components such as polysaccharides and proteins, as well as the retention rate of Rubescensine A. Test method:

[0124] Preparation of test solution: 15 g of Shenxianling granule powder was taken and the test solution was prepared according to the method of Example 1, and divided into two groups:

[0125] Experimental group: 1.5 g of composite impurity remover (formula of Example 1) was added;

[0126] Control group: no compound impurity remover was added.

[0127] Index detection:

[0128] Polysaccharide content: The phenol-sulfuric acid method was used with glucose as the reference substance, and the absorbance was measured at a wavelength of 490 nm.

[0129] Protein content: The Coomassie brilliant blue method was used to measure the absorbance at a wavelength of 595 nm with bovine serum albumin as the reference.

[0130] Oridonin retention rate: The concentration of Oridonin before and after treatment was determined by HPLC, and the retention rate was calculated.

[0131] Test data:

[0132] Table 3

[0133] Group Polysaccharide removal rate (%) Protein removal rate (%) Oridonin retention rate (%) Experimental group 92.3±1.5 87.6±2.1 98.5±0.8 control group 12.7±3.2 15.4±4.3 99.2±0.5

[0134] Conclusion: The removal rate of the composite impurity remover for macromolecular impurities such as polysaccharides and proteins was significantly higher than that of the untreated group (p<0.01), and there was no obvious adsorption loss of Rubescensine A, which proved that it could effectively retain the target components while purifying the sample and improve the detection accuracy.

[0135] Figure 1 The figure is a curve diagram showing the effect of different composite adsorbent calcination temperatures on their polysaccharide removal rates, indicating that a higher polysaccharide removal rate can be maintained within the calcination temperature range of 400-430°C.

[0136] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification.

Claims

1. A method for determining and identifying the content of Shenxianling granules, characterized in that: It includes the use of high-performance liquid chromatography (HPLC) to determine the content of oridonin in Shenxianling granules, and the use of thin-layer chromatography (TLC) to identify six ingredients, including herb agrimony, black plum, liquorice, Gynostemma pentaphyllum, ginseng, and Ganoderma lucidum. The TLC identification method for each ingredient includes a unique preparation method for the test solution, control medicinal material solution, and negative control solution, as well as the specific settings of the sample spot volume, developing agent, and inspection conditions.

2. The method according to claim 1, characterized in that The chromatographic conditions for the HPLC method for determining the content of Rubescensine A are: Phenomenon C18 chromatographic column (250 mm×4.6 μm, 5 μm); mobile phase of methanol:water = 65:35; flow rate 0.8-1.0 ml / min; column temperature 25-26° C.; detection wavelength 238 nm; injection volume 20 μl.

3. The method according to claim 1, characterized in that The preparation and pretreatment method of the test solution is as follows: grind the Shenxianling granules, weigh 15-16g and place them in a Soxhlet extractor, add 100ml of petroleum ether, heat and reflux at 60-90°C for 2.5h, discard the petroleum ether, evaporate the petroleum ether, add 30ml of methanol and soak for 2-3h, ultrasonicate for 20-30min, pour out the methanol extract, and then use 20ml of methanol to operate 5 times in the same way, combine the methanol extracts and evaporate to dryness, dilute the residue to a 50ml volumetric flask with a solution of methanol: water = 65:35, add 1.5-1.8g of a composite impurity remover, shake for 15-20min, filter, and filter the filtrate with a 0.4μm microporous filter membrane to obtain the test solution; The composite impurity remover is prepared from silica gel-alumina-activated carbon through a cross-linking-calcination process; The composite impurity remover is prepared by mixing silica gel (particle size 50-100 μm), neutral alumina, and activated carbon in a mass ratio of 3-5:2-3:1-2, adding a 5-6% polyvinyl alcohol aqueous solution as a cross-linking agent, stirring at 60° C. into a paste, extruding into particles with a diameter of 1-2 mm, calcining in a muffle furnace at 400-430° C. for 2-3 hours, and filtering through an 80-mesh sieve after cooling for later use; The mass fraction of the polyvinyl alcohol aqueous solution is 20%.

4. The method according to claim 1, wherein The preparation method of the reference solution is as follows: accurately weigh 8 mg of oridonin standard, add 4 ml of methanol to dissolve it to prepare a 2 mg / ml oridonin stock solution; when preparing the standard curve, dilute the oridonin stock solution with methanol to obtain reference solutions with concentrations of 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, and 0.78 μg / ml, respectively; dispense the solutions into 1.5 ml centrifuge tubes, and store at 4°C for future use.

5. The method according to claim 1, wherein When the TLC method is used to identify the components of Agrimoniae: Preparation of test solution: Take 5 g of Agrimony Root, add 60 ml of petroleum ether (60-90 ℃) and ultrasonically treat for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of chloroform, shake and extract with 10 ml of 5% sodium hydroxide solution, discard the chloroform solution, adjust the pH value of the sodium hydroxide solution to 1-2 with dilute hydrochloric acid, and then shake and extract with chloroform twice (10 ml each time), combine the chloroform solutions, add 10 ml of water to wash, discard the water solution, and concentrate the chloroform solution to 1 ml; Control system: 5 g of Agrimoniae negative preparation and 0.5 g of Agrimoniae control medicinal material were used to prepare negative control solution and control medicinal material solution in the same manner; Spotting and development: Pipette 10 μl each of the test solution, negative control solution and control medicinal material solution, spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of petroleum ether (60-90℃)-ethyl acetate-acetic acid (100:9:5) as the developing agent.

6. The method according to claim 1, characterized in that When the TLC method is used to identify the components of black plum: Preparation of test solution: Take 15g of the powder of this product, add 40ml of methanol and sonicate for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, add ether and shake to extract twice (20ml each time), combine the ether solution and evaporate to dryness, soak the residue in petroleum ether (30-60℃) twice (20ml each time), pour off the petroleum ether, and dissolve the residue in 1ml of anhydrous ethanol; Control system: 15g of Wumei negative preparation and 1g of Wumei control medicinal material. Prepare negative control solution and control medicinal material solution in the same way; Spotting and development: 5 μl of the test solution, 5 μl of the negative control solution, and 10 μl of the control medicinal material solution were applied to the same silica gel G thin layer plate and developed with cyclohexane-chloroformane-ethyl acetate-formic acid (20:5:8:0.1) as the developing solvent.

7. The method according to claim 1, characterized in that When the TLC method is used to identify the components of licorice: Preparation of test solution: Take 15g of the powder of this product, add 60ml of ether and heat under reflux for 1h, filter and discard the ether solution, add 60ml of methanol to the residue and heat under reflux for 1h, filter, evaporate the filtrate to dryness, dissolve the residue in 40ml of water, extract with n-butanol 3 times (20ml each time), combine the n-butanol solutions, wash 3 times with water, discard the water solution, evaporate the n-butanol solution to dryness, and dissolve the residue in 1ml of methanol; Control system: 15g of negative preparation lacking licorice and 1g of licorice control medicinal material were used to prepare negative control solution and control medicinal material solution in the same way; Spotting and development: Pipette 10 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, and develop with ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing agent.

8. The method according to claim 1, characterized in that When the TLC method is used to identify the components of Gynostemma pentaphyllum: Preparation of test solution: Take 15g of the powder of this product, add 60ml of methanol and sonicate for 1h, cool and filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract with saturated n-butanol 3 times (20ml each time), combine the n-butanol solutions and evaporate to dryness, dissolve the residue in 1ml of methanol; Control system: 15 g of the negative preparation lacking Gynostemma pentaphyllum and 1 g of the Gynostemma pentaphyllum control medicinal material were used to prepare the negative control solution and the control medicinal material solution in the same manner; Spotting and development: Pipette 5μl of the test solution and negative control solution, and 10μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, and develop with the upper layer solution of n-butanol-ethyl acetate-water (4:1:5) placed below 10℃ as the developing agent.

9. The method according to claim 1, characterized in that When the TLC method is used to identify ginseng components: Preparation of test solution: Take 15g of the powder of this product, add 100ml of chloroform and heat under reflux for 1h, discard the chloroform solution, evaporate the solvent from the residue, add 1.0ml of water and stir to moisten it, add 30ml of water-saturated n-butanol and ultrasonicate for 1h, aspirate the supernatant, add 3 times the amount of ammonia test solution, shake well and let it separate, take the upper layer and evaporate to dryness, and dissolve the residue in 1ml of methanol; Control system: 15g of ginseng negative preparation and 1g of ginseng control medicinal material were used to prepare negative control solution and control medicinal material solution in the same way; Spotting and development: Pipette 10 μl each of the test solution, negative control solution, and control medicinal material solution, and spot them on the same silica gel G thin layer plate. Develop with the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) stored below 10°C as the developing agent.

10. The method according to any one of claims 1 to 9, characterized in that When the TLC method is used to identify the components, the samples must be air-dried after development, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots are clearly colored, and inspected under ultraviolet light (365nm); in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, while there are no spots at the corresponding positions in the negative control solution.

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